The p21-activated protein kinases (Paks) are to be key effectors for Cdc42 and Rac1; two Rho-family GTPases that regulate a variety of fundamental biological processes, including cell proliferation, shape control, migration, and stress response. Abnormalities in these processes underlie many important human diseases, including most malignancies. Recently, we, and others, have implicated Paks in two specific processes that are germane to tumorigenesis: genomic stability (via regulation of centrosome function) and cell proliferation (via effects on elements in the ERK signal transduction cascade). These findings suggest that Paks play a central role in tumorigenesis and that these enzymes might be suitable targets for anti- neoplastic therapy. Here, we seek to uncover the mechanisms by which Paks regulate these vital processes in breast epithelial cells and if these mechanisms also apply in living organisms. In the first aim, we will use both gain-of-function and loss-of-function methods to study the role of Pak in genomic stability, with particular emphasis on its effects on the centrosome and mitotic spindle. In the second aim, we will use both biochemical and genetic means to examine how group A Paks regulate the ERK signaling pathway in mammary epithelial cells grown in a three-dimensional format. The use of a specific Pak inhibitor, as well as cells derived from our recently constructed Pak1 and Pak2 knock-out mice, give us unique reagents with which to accomplish these goals. In the third aim, we will test if Pak function is required for tumorigenesis in a breast cancer model, using Neu-transgenic mice crossed with our Pak knock-out mice and also with a transgenic mouse that expresses a specific Pak inhibitor in mammary tissues. Achieving the aims set forth in this proposal will shed light on the mechanisms by which Paks regulate two fundamental biologic properties that are germane to human tumorigenesis: genomic stability and cell proliferation. For these reasons, understanding Pak function is not only of intrinsic scientific interest but is also likely to be relevant to identifying useful new targets for future cancer therapy. The proposed work is directly relevant to our understanding and treatment of breast cancer. If we establish that p21-activated kinases (Paks) are required for the Neu2 oncogene (one of the most commonly mutated genes in human breast cancer) to cause breast cancer in mice, then drugs that block Pak function might be used as a new and specific means to treat this disease in humans. [unreadable] [unreadable] [unreadable]